Recognition of the replication origin by initiator protein is the central event regulating DNA replication in diverse biological systems. The long term objective of this research is to understand the initiation process using the gamma origin replicon of the antibiotic resistance plasmid R6K as a model system. The R6K-encoded pi protein regulates replication and autoregulates its own expression by binding to DNA sites containing a TGARG sequence motif arranged either into seven 22-bp direct repeats (DRs) in the gamma ori, or into inverted half-repeats in the operator of its own gene pir. Pi protein has a modular structure; the C-terminus contains the DNA binding domain while the N-terminus controls pi oligomerization. The model of pi protein activity proposes that its distinct surfaces facilitate assemblies on DRs and IRs. Experiments described in Specific Aim #1 test this model using available variants of pi protein that differ from wild type pi in the control of replication and transcription. DNA and protein footprinting, photo-crosslinking and genetic fusion techniques will be employed in these investigations. In Specific Aim #2 the investigators will identify the nucleotides in pi binding sites and the amino acids in pi protein that intimately contact one another by using missing nucleotide technology and by genetically altering the DNA binding properties of pi. The investigators have recently identified a novel, non-TGAGRG pi binding site in the A+T-rich segment of gamma ori and have mapped the start of DNA synthesis approximately 30-bp upstream from this site. Specific Aim #3 will define the relationship between pi binding to this novel site and initiation by using an available in vitro replication system. The priming reaction will also be reconstituted with purified components and the effect of pi will be examined in this system. The proposed studies will reveal at a molecular level the mechanisms by which a single regulatory protein independently regulates replication and transcription.